10 research outputs found
Cytochrome P450-mediated bioactivation of mefenamic acid to quinoneimine intermediates and inactivation by human glutathione S-transferases
Mefenamic acid (MFA) has been associated with rare but severe cases of hepatotoxicity, nephrotoxicity, gastrointestinal toxicity, and hypersensitivity reactions that are believed to result from the formation of reactive metabolites. Although formation of protein-reactive acylating metabolites by phase II metabolism has been well-studied and proposed to be the cause of these toxic side effects, the oxidative bioactivation of MFA has not yet been competely characterized. In the present study, the oxidative bioactivation of MFA was studied using human liver microsomes (HLM) and recombinant human P450 enzymes. In addition to the major metabolite 3′-OH-methyl-MFA, resulting from the benzylic hydroxylation by CYP2C9, 4′-hydroxy-MFA and 5-hydroxy-MFA were identified as metabolites resulting from oxidative metabolism of both aromatic rings of MFA. In the presence of GSH, three GSH conjugates were formed that appeared to result from GSH conjugation of the two quinoneimines formed by further oxidation of 4′-hydroxy-MFA and 5-hydroxy-MFA. The major GSH conjugate was identified as 4′-OH-5′-glutathionyl-MFA and was formed at the highest activity by CYP1A2 and to a lesser extent by CYP2C9 and CYP3A4. Two minor GSH conjugates resulted from secondary oxidation of 5-hydroxy-MFA and were formed at the highest activity by CYP1A2 and to a lesser extent by CYP3A4. Additionally, the ability of seven human glutathione S-transferases (hGSTs) to catalyze the GSH conjugation of the quinoneimines formed by P450s was also investigated. The highest increase of total GSH conjugation was observed with hGSTP1-1, followed by hepatic hGSTs hGSTA2-2 and hGSTM1-1. The results of this study show that, next to phase II metabolites, reactive quinoneimines formed by oxidative bioactivation might also contribute to the idiosyncratic toxicity of MFA. (Chemical Presented)
Application of engineered cytochrome P450 mutants as biocatalysts for the synthesis of benzylic and aromatic metabolites of fenamic acid NSAIDs
Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme
In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with >95 % regioselectivity to form 16-β-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-β-hydroxylated product, resulting in the formation of about 0.35 g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions
Cytochrome P450-Mediated Bioactivation of Mefenamic Acid to Quinoneimine Intermediates and Inactivation by Human Glutathione S‑Transferases
Mefenamic acid (MFA) has been associated
with rare but severe cases
of hepatotoxicity, nephrotoxicity, gastrointestinal toxicity, and
hypersensitivity reactions that are believed to result from the formation
of reactive metabolites. Although formation of protein-reactive acylating
metabolites by phase II metabolism has been well-studied and proposed
to be the cause of these toxic side effects, the oxidative bioactivation
of MFA has not yet been competely characterized. In the present study,
the oxidative bioactivation of MFA was studied using human liver microsomes
(HLM) and recombinant human P450 enzymes. In addition to the major
metabolite 3′-OH-methyl-MFA, resulting from the benzylic hydroxylation
by CYP2C9, 4′-hydroxy-MFA and 5-hydroxy-MFA were identified
as metabolites resulting from oxidative metabolism of both aromatic
rings of MFA. In the presence of GSH, three GSH conjugates were formed
that appeared to result from GSH conjugation of the two quinoneimines
formed by further oxidation of 4′-hydroxy-MFA and 5-hydroxy-MFA.
The major GSH conjugate was identified as 4′-OH-5′-glutathionyl-MFA
and was formed at the highest activity by CYP1A2 and to a lesser extent
by CYP2C9 and CYP3A4. Two minor GSH conjugates resulted from secondary
oxidation of 5-hydroxy-MFA and were formed at the highest activity
by CYP1A2 and to a lesser extent by CYP3A4. Additionally, the ability
of seven human glutathione S-transferases (hGSTs) to catalyze the
GSH conjugation of the quinoneimines formed by P450s was also investigated.
The highest increase of total GSH conjugation was observed with hGSTP1-1,
followed by hepatic hGSTs hGSTA2-2 and hGSTM1-1. The results of this
study show that, next to phase II metabolites, reactive quinoneimines
formed by oxidative bioactivation might also contribute to the idiosyncratic
toxicity of MFA
Human NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated inactivation of reactive quinoneimine metabolites of diclofenac and mefenamic acid
NAD(P)H: quinone oxidoreductase 1 (NQO1) is an enzyme capable of reducing a broad range of chemically reactive quinones and quinoneimines (QIs) and can be strongly upregulated by Nrf2/Keap1-mediated stress responses. Several commonly used drugs implicated in adverse drug reactions (ADRs) are known to form reactive QI metabolites upon bioactivation by P450, such as acetaminophen (APAP), diclofenac (DF), and mefenamic acid (MFA). In the present study, the reductive activity of human NQO1 toward the QI metabolites derived from APAP and hydroxy-metabolites of DF and MFA was studied, using purified bacterial P450 BM3 (CYP102A1) mutant M11 as a bioactivation system. The NQO1-catalyzed reduction of the QI metabolites was quantified relative to spontaneous glutathione (GSH) conjugation. Addition of NQO1 to the incubations strongly reduced the formation of all corresponding GSH conjugates, and this activity could be prevented by dicoumarol, a selective NQO1 inhibitor. The GSH conjugation was strongly increased by adding human GSTP1-1 in a wide range of GSH concentrations. Still, NQO1 could effectively compete with the GST catalyzed GSH conjugation by reducing the QIs. In conclusion, we identified the QI metabolites of the 4'- and 5-hydroxy-metabolites of DF and MFA as novel substrates for human NQO1. NQO1-mediated reduction proves to be an effective pathway to detoxify these QI metabolites in addition to GSH conjugation. Genetically determined deficiency of NQO1 therefore might be a risk factor for ADRs induced by reactive QI drug metabolites
Empirical Study on Sustainable Opportunities Recognition. A Polyvinyl Chloride (PVC) Joinery Industry Analysis Using Augmented Sustainable Development Process Model
Surface-Modified Shortwave-Infrared-Emitting Nanophotonic Reporters for Gene-Therapy Applications
Gene
therapy is emerging as the next generation of therapeutic
modality with United States Food and Drug Administration approved
gene-engineered therapy for cancer and a rare eye-related disorder,
but the challenge of real-time monitoring of on-target therapy response
remains. In this study, we have designed a theranostic nanoparticle
composed of shortwave-infrared-emitting rare-earth-doped nanoparticles
(RENPs) capable of delivering genetic cargo and of real-time response
monitoring. We showed that the cationic coating of RENPs with branched
polyethylenimine (PEI) does not have a significant impact on cellular
toxicity, which can be further reduced by selectively modifying the
surface characteristics of the PEI coating using counter-ions and
expanding their potential applications in photothermal therapy. We
showed the tolerability
and clearance of a bolus dose of RENPs@PEI in mice up to 7 days after
particle injection in addition to the RENPs@PEI ability to distinctively
discern lung tumor lesions in a breast cancer mouse model with an
excellent signal-to-noise ratio. We also showed the availability of
amine functional groups in the collapsed PEI chain conformation on
RENPs, which facilitates the loading of genetic cargo that hybridizes
with target gene in an in vitro cancer model. The real-time monitoring
and delivery of gene therapy at on-target sites will enable the success
of an increased number of gene- and cell-therapy products in clinical
trials